Ten years ago, the food and drug administration first approved the use of plasminogen activators for thrombolytic therapy. It was originally recommended for the treatment of deep-vein thrombosis and serious pulmonary embolisms. This approach is now also used for treating acute peripheral arterial thrombosis and acute coronary thrombosis and for solubilizing clots in catheters and shunts.
With the development of recombinant DNA technology and the cloning and expression of tissue plasminogen activator (TPA), we have now entered a new age for thrombolytic agents with unique physiological properties and therapeutic promise. The original plasminogen activators that have been used clinically were streptokinase and urokinase. These agents produced in the patients a generalized lytic state which had a variety of side effects that were not directly targeted at solubilizing the fibrin clot. Tissue plasminogen activator, because of its fibrin binding capacity, enhances the selectivity of these agents for fibrin degradation. It is thus being greeted with great fanfare as the new generation of fibrinolytic agents.
Tissue plasminogen activator is not without its potential side effects, and, furthermore, its cost is prohibitive for use in many settings. For example, the V.A. in Gainesville will not authorize tissue plasminogen activator therapy over streptokinase therapy because of its enormous cost. Furthermore, the application of this fibrinolytic therapy to domestic animals or food production animals is limited by the enormous cost.
It is clear to most experts that the wonder drug nature of tissue plasminogen activator has been highly overrated. TPA has been found to have a very short half life in the body. Also, pharmacological doses of tissue plasminogen activator produce a significant bleeding risk for the patient, and in the case of coronary artery thrombosis, re-occlusion of the blood vessel following successful clot lysis occurs in a significant number of patients. The need for an inexpensive, and perhaps even better form of treatment is clearly evident. The use of combination therapies whereby existing compositions and methods are integrally linked in novel ways with new materials and procedures could enhance the effectiveness of plasminogen activators and possibly reduce the amount of plasminogen activator needed to achieve the desired results.
Recently, we have described in our laboratory the presence of a selective, high affinity receptor for human plasmin and other species of plasmin on the surface of certain group A streptococci (Lottenberg, R., C. C. Broder, M. D. P. Boyle [1987] Infect. Immun. 55:1914-1918). Group A streptococci cause pharyngitis and invasive infections such as cellulitis and bacteremia (Bisno, A. L. [1991] New Engl. J. Med. 325:783-793). There has been a recent increase in invasive group A streptococcal infections occurring in healthy individuals. Also, group B streptococci are important pathogens for pregnant women. Staphylococcus aureus, another gram-positive bacteria, is another invasive pathogen for immunocompetent hosts. Effective vaccines to protect against infection by these organisms are not currently available.
The biochemical interactions occurring at cell surfaces between bacterial membranes and their surroundings are complex and not well understood. Certain bacterial surface structures and secreted products have been suggested to contribute to tissue invasion. One of these secreted products, streptokinase, is a plasminogen activator and converts the host zymogen plasminogen to the active protease, plasmin (Siefring, G. E., F. J. Castellino [1976] J. Biol. Chem. 251:3913-3920). Although classically described as the enzyme responsible for fibrin degradation, plasmin is a serine protease with trypsin-like specificity and has activity for a broad range of substrates. Plasmin can degrade several mammalian extracellular matrix proteins, such as fibronectin and laminin, and can enhance collagenase activity (Liotta, L. A., R. H. Goldfarb, R. Brundage [1981] Cancer Res. 41:4629-4636). Therefore, the ability to generate and capture active plasmin may contribute to the invasive propensity of certain streptococcal strains. The interaction of plasmin with group A streptococci has high affinity (K.sub.d, 10.sup.-10 M) and is specific for plasmin, with no significant binding demonstrated for structurally related proteins (Broeseker, T. A., M. D. P. Boyle, R. Lottenberg [1988] Microb. Pathog. 5:19-27; DesJardin, L. E., M. D. P. Boyle, R. Lottenberg [1989] Thromb. Res. 55:187-193).
As described herein, the plasmin receptor of the subject invention has significant similarity to the glycolytic enzyme glyceraldehyde 3-phosphate dehydrogenase (GAPDH). GAPDH is a key enzyme involved in glucose metabolism and has been the subject of many genetic studies. Multiple copies of GAPDH genes have been identified for mammals, with many described as pseudogenes (Piechaczyk, M., J. M. Blanchard, S. Riaad-El Sabouty, C. Dani, L. Marty, P. Jeanteur [1984] Nature 312:469-471). Multiple GAPDH genes have also been identified for E. coli, Tiypanosoma brucei, Saccharomyces cerevisiae, and Drosophila melanogaster (Alefounder, P. R., R. N. Perham [1989] Mol. Microbiol. 3:723-732; Holland, J. P., L. Banieniec, C. Swimmer, M. J. Holland [1983] J. Biol. Chem. 258:5291-5299; Michels, P. A. M., M. Marchand, L. Kohl, S. Allert, R. K. Wierenga, F. R. Opperdoes [1991] Eur. J. Biochem. 198:421-428; Tso, J. Y., X. -H. Sun, R. Wu [1985] Nucleic Acids Res. 17:1251). E. coli and T. brucei each have two GAPDH genes with significant differences in deduced amino acid sequence (Alefounder et al., supra; Michels et al., supra); however, the translated product of the second E. coli GAPDH gene has not been reported. One of the trypanosomal isoenzymes is localized in the glycosome, a specialized metabolic organelle, while the other GAPDH is found in the cytoplasm (Lambier, A. -M., A. M. Loiseau, D. A. Kuntz, F. M. Vellieux, P. A. M. Michels, F. R. Opperdoes [1991] Eur. J. Biochem. 198:429-435).
In addition to its usual intracellular location, GAPDH has been identified on the surface of hematopoietic cells and Schistosoma mansoni, an invasive parasite (Allen, R. W., K. A. Trach, J. A. Hoch [1987] J. Biol. Chem. 262:649-653; Goudot-Crozel, V., D. Caillol, M. Djabali, A. J. Dessein [1989] J. Exp. Med. 170:2065-2080). Allen and Hoover ([1985] Blood 65:1045-1055) characterized a membrane-associated 37,000-M.sub.r protein expressed by the erythroleukemic cell line K562. Peptide mapping and molecular cloning studies revealed the protein to be homologous to GAPDH (Allen, Trach, and Hoch, supra). A similar finding has been reported for the blood fluke responsible for abdominal schistosomiasis (Goudet-Crozel et al., supra). A 37,000-M.sub.r surface immunogen of S. mansoni was characterized by isolating the cDNA encoding the protein. The deduced amino acid sequence had significant homology to that of human GAPDH. Like the recombinant plasmin receptor protein (Plr), neither of these surface proteins had domains corresponding to previously described membrane-anchoring structures (Blobel, G. [1980] Proc. Natl. Acad. Sci. USA 77:1496-1500; Ferguson, M. A. J., A. F. Williams [1988] Annu. Rev. Biochem. 57:285-320). Interestingly, Hekman et al. (Hekman, W. E., D. T. Dennis, J. A. Miemyk [1990] Mol. Microbiol. 4:1363-1369), while studying the expression of recombinant plant GAPDH in E. coli, were able to target the protein to the outer membrane by genetically fusing the signal sequence of E. coli OmpA to Ricinus communis GAPDH. Pancholi et al. have recently reported the isolation of a 39 kD surface protein with GAPDH activity from a group A streptococci (Pancholi, V., V. A. Fischetti [1992] "A Novel Multifunctional Surface Protein (MFG) of group A Streptococci," Abstract No. B-252, Abstracts of the General Meeting 1992:68).
GAPDH from streptococci has not been isolated or characterized, and the relationship of the plasmin receptor to the glycolytically active enzyme remains to be seen.